System and method to measure tissue biomechanical properties without external excitation

ABSTRACT

A system and method for measuring biomechanical properties of tissues without external excitation are capable of measuring and quantifying these parameters of tissues in situ and in vivo. The system and method preferably utilize a phase-sensitive optical coherence tomography (OCT) system for measuring the displacement caused by the intrinsic heartbeat. The method allows noninvasive and nondestructive quantification of tissue mechanical properties. Preferably, the method is used to detect tissue stiffness and to evaluate its stiffness due to intrinsic pulsatile motion from the heartbeat. This noninvasive method can evaluate the biomechanical properties of the tissues in vivo for detecting the onset and progression of degenerative or other diseases and evaluating the efficacy of therapies.

This application is a continuation of and claims priority to U.S. patentapplication Ser. No. 16/907,424, filed Jun. 22, 2020, entitled “Systemand Method to Measure Tissue Biomechanical Properties Without ExternalExcitation,” which is a continuation-in-part of and claims priority toU.S. patent application Ser. No. 16/558,413, filed Sep. 3, 2019,entitled “Optical Coherence Elastography to Assess Biomechanics andDetect Progression of Ocular and Other Tissues Degenerative Diseases,”which is a divisional of and claims priority to U.S. patent applicationSer. No. 15/632,657, filed Jun. 26, 2017, entitled “Optical CoherenceElastography to Assess Biomechanics and Detect Progression of Ocular andOther Tissues Degenerative Diseases,” which is a continuationapplication of and claims priority to U.S. patent application Ser. No.14/934,663, filed Nov. 6, 2015, entitled “Optical Coherence Elastographyto Assess Biomechanics and Detect Progression of Ocular and OtherTissues Degenerative Diseases,” which claims priority to U.S.Provisional Patent Application Ser. No. 62/077,561, filed Nov. 10, 2014,entitled “Optical Coherence Elastography to Detect the Onset andProgression of Corneal Degenerative Diseases,” and U.S. ProvisionalPatent Application Ser. No. 62/171,043, filed Jun. 4, 2015, entitled“Ultrasound and Optical Coherence Elastography to Assess Biomechanics ofOcular and Other Tissues,” and the entire contents of these applicationsare hereby incorporated by reference.

The present invention used in part funds from the National Institute ofHealth (NIH), No. R01EY022362. The United States Government has certainrights in the invention.

BACKGROUND

This disclosure pertains to a method for assessing the biomechanicalproperties of the cornea and other tissues and for detecting anddifferentiating tissue stiffness without the need for any externalexcitation. The pulsatile motion within the body, e.g., from theheartbeat, creates small displacements, which are detected by a system.

Changes in the viscoelastic properties of tissues are associated withthe onset and progression of different diseases as well as changes dueto therapeutic procedures. Therefore, it is essential to assess andquantify tissue mechanical properties for disease detection, duringdisease progression, and for evaluation of different therapeuticprocedures.

For example, keratoconus is associated with localized reduced rigidityof the cornea, and the information regarding corneal stiffness is usefulto provide improved diagnosis and monitoring of this pathologicalstatus. Also, real-time in vivo measurement of the spatial elasticitydistribution with microscopic scale in the cornea could lead to adaptivemechanical modeling of the individual corneal structure which isextremely important to prevent over-corrections, under-corrections, andectasia from refractive surgeries, such as LASIK, and to furtheroptimize surgical procedures.

Structurally degenerative diseases such as keratoconus can significantlyalter the stiffness of the cornea, directly affecting the quality ofvision. Keratoconus can pathologically decrease the localized stiffnessof the cornea, leading to a loss in the quality of vision. Detectingchanges in the biomechanical properties of ocular tissues, such asstiffness of the cornea, can aid in the diagnosis of these structurallydegenerative diseases.

UV-induced collagen cross-linking (CXL) is an emerging treatment thateffectively increases corneal stiffness and is applied clinically totreat keratoconus. The effectiveness of this treatment may be analyzedand improved by measuring the corneal stiffness both before and aftertreatment.

In addition, cardiovascular and cerebrovascular diseases and associatedischemic events can be caused by changes in vasculature stiffness.Hardening of the vessel wall increases blood pressure because thevessels are less compliant, and therefore, cannot accommodate during thepulsatile activity during blood pulsation. Therefore, measuring thestiffness of the vasculature can provide critical information forcardiovascular health.

Elastography is an emerging technique that can map the local mechanicalproperties of tissues. Ultrasound elastography (USE) and magneticresonance elastography (MRE) have experienced rapid development duringthe past couple of decades as clinical diagnostic tools. One commonprinciple of these techniques is correlating tissue deformation causedby the external mechanical excitation to tissue elasticity. However,these techniques' use of external excitation limits evaluation of thetissue mechanical properties due to various factors such as excitationbandwidth, tissue response, and patient comfort. The basic feasibilityof using Brillouin microscopy to measure the cornea elasticity both invitro and in vivo has been explored. Brillouin microscopy can beimplemented using simple instrumentation, but it has a relatively slowacquisition time. There is also uncertainty on how to correlateBrillouin shift (modulus) to the classical mechanical description oftissues (e.g. Young modulus). Other nano-scale elastography techniques,such as atomic force microscopy, require lengthy acquisition times andcontact with the tissue.

What is needed, therefore, is an improved, noninvasive and highlysensitive method to assess the mechanical properties of the ocular andother tissues with high resolution and sensitivity, preferably withoutcontact with the tissue.

Moreover, measuring the stiffness of the cornea, or other oculartissues, without any external excitation, would further improve thecapability for ocular tissue assessment by ensuring there are no issueswith patient discomfort and for even more rapid imaging.

SUMMARY

The present disclosure relates generally to methods and systems forassessing the biomechanical properties of tissues non-invasively, to amethod using, for example, optical coherence elastography (OCE), fordetecting tissue stiffness, such as stiffness of the cornea, sclera,skin, blood vessels, and the like. The methods described herein forbiomechanical tissue quantification are demonstrated in the case of thecornea but are generally applicable for all soft and hard tissues in thebody.

Optical coherence elastography (OCE) is capable of direct andhigh-resolution assessment of mechanical properties of tissue and,therefore, overcomes the limitations of previously-used techniques. OCEgenerally employs high-resolution optical coherence tomography (OCT) todetect the sample deformation induced by an external force. Incomparison to ultrasound elastography (USE) and magnetic resonanceelastography (MRE), OCE can provide superior spatial imaging resolution,faster acquisition speed, and greater displacement sensitivity.

In one aspect, this disclosure relates to a method for quantifyingbiomechanical properties of a tissue, comprising: using opticalcoherence tomography (OCT) or any other low-coherence interferometryand/or phase-sensitive subsystem to image a tissue sample and measureintrinsic displacements, generally produced by the pulsatile motion fromthe cardiovascular system; and quantifying the biomechanical propertiesof the tissue based on the analysis of the displacement. The step ofquantifying may use an algorithm.

The present system utilizes a phase-sensitive OCT system for measuringthe tissue displacement caused by the intrinsic pulses from thepulsatile forces of the cardiovascular system. The system allows for anoninvasive and highly sensitive method to assess the mechanicalproperties of the tissue in vivo.

The present method compares the displacement amplitude induced by thepulsatile forces from the cardiovascular system. The amplitude of thedisplacement is mapped and compared at different states during thepulsatile motion. This noninvasive method has the potential to detectthe early stages of ocular diseases such as keratoconus or to be appliedduring cross-linking (CXL) procedures for therapy evaluation andpersonalization.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a flow chart describing an exemplary method for imagingpulsatile motion in tissue, such as the cornea, induced by an intrinsicpulse, such as a heartbeat, during imaging, and quantifying tissuebiomechanical properties.

FIG. 2 shows a schematic of an exemplary phase-sensitive opticalcoherence elastography (OCE) system for measuring corneal displacementdue to a simulated ocular pulse in an in situ cornea sample in the wholeeye-globe configuration. CCD: charged coupled device, DAC: digital toanalog converter, IOP: intraocular pressure, SLD: superluminescentdiode.

FIG. 3 shows (top left) instantaneous displacement in an untreated insitu porcine cornea, where the fluid is infused into the eye-globe tosimulate one phase of the pulsatile motion in the cornea; (bottom left)instantaneous displacement in an untreated in situ porcine cornea wherefluid is withdrawn from the eye-globe to simulate one phase of thepulsatile motion in the cornea; (top right) instantaneous displacementin an UV/riboflavin crosslinked in situ porcine cornea where fluid isinfused into the eye-globe to simulate one phase of the pulsatile motionin the cornea, (bottom right) instantaneous displacement in anUV/riboflavin crosslinked in situ porcine cornea where fluid iswithdrawn from the eye-globe to simulate one phase of the pulsatilemotion in the cornea.

FIG. 4 shows (top left) strain in an untreated in situ porcine corneawhere fluid is infused into the eye-globe to simulate one phase of thepulsatile motion in the cornea, (bottom left) strain in an untreated insitu porcine cornea where fluid is withdrawn from the eye-globe tosimulate one phase of the pulsatile motion in the cornea, (top right)strain in an UV/riboflavin crosslinked in situ porcine cornea wherefluid is infused into the eye-globe to simulate one phase of thepulsatile motion in the cornea, (bottom right) strain in anUV/riboflavin crosslinked in situ porcine cornea where fluid iswithdrawn from the eye-globe to simulate one phase of the pulsatilemotion in the cornea.

FIG. 5 shows the strain in an in situ porcine cornea (triangle) before(UT) and (square) after UV/riboflavin crosslinking (CXL) plottedalongside the intraocular pressure (IOP) of the eye-globe.

FIG. 6 shows (a) plots of the average and standard deviation of 3 insitu porcine corneas (triangle) before (UT) and (square) after (CXL)UV/riboflavin crosslinking plotted alongside the (solid line) eye-globeintraocular pressure (IOP) and (b) stiffness of the porcine corneas (UT)before and (CXL) after UV/riboflavin crosslinking with the p-value ofthe two sample t-test indicated.

FIG. 7 shows (a) a structural OCT image of a partially UV/riboflavincrosslinked in situ porcine cornea where the left side was crosslinked(CXL) and the right side was untreated (UT) and (b) the localized strainin both regions (triangle—untreated; square—CXL) plotted alongside the(solid line) intraocular pressure (IOP) within the eye-globe.

FIGS. 8 (a) and (b) show sample displacement frames and correspondingvoltage from a chest-mounted pressure transducer during in vivo imagingof an anesthetized rabbit. The cornea was compressed against a glassslide for stabilization. The frame time is indicated by the verticalline in the transducer voltage plot in the bottom sub-figure.

FIG. 8 (c) shows a profile of the average corneal strain plottedalongside the voltage of the chest-mounted pressure transducer showingthat the present method can measure the pulsatile motion in the corneadue to the heartbeat in vivo.

FIG. 9 shows (a) the (line) average and (shaded region) standarddeviation of the strain in a rabbit cornea in vivo (UT, dashed line)before and (CXL, solid line) after riboflavin/UV crosslinking and (b)strain after crosslinking, indicating a significant increase instiffness.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present disclosure relates to methods utilizing optical coherenceelastography (OCE) to detect tissue stiffness with no externalexcitation. Previous studies have demonstrated that OCE is feasible forquantitatively assessing the elasticity of a sample but requiredexternal excitation. Preferably, the method is used to quantify cornealbiomechanical properties. The present method uses phase-sensitive OCTand can distinguish untreated (UT) and riboflavin/UV cross-linked (CXL)corneas due to differences in stiffness and differences in stiffness dueto different baseline intraocular pressures. The method can furtherdifferentiate UT and C×L regions in a partially crosslinked cornea. Thisnoninvasive method can evaluate the biomechanical properties of thecornea in vivo for detecting the onset and progression of cornealdegenerative diseases such as keratoconus and for evaluating theefficacy of therapies such as CXL.

FIG. 1 shows a flow chart illustrating an example of a methodencompassed by the present disclosure. The sample can be stabilized andis imaged rapidly by the phase-sensitive optical coherence elastography(PhS-OCE) system. An electrocardiogram can be measured for corroboratingthe OCE-measured data with the heartbeat. The phase difference iscalculated from successive OCT frames after motion correction, which isthen translated to displacement. Since the structural image is alsoobtained, the strain is then mapped on to the cornea. Either with orwithout external IOP measurement, the strain can then be converted toelasticity.

Generally, the present method is for measuring or assessing tissuebiomechanical properties (e.g. stiffness and viscosity) with no externalexcitation. In an exemplary embodiment, a first step is imaging thetissue sample at one or more regions of the tissue sample using phasesensitive and sufficiently high speed interferometry (low-coherenceinterferometry or optical coherence tomography, which is preferred). Thesample may be imaged in either 2D (axial and transverse), 3D (axial andboth transverse dimensions), or 4D (axial, both transverse axes, andtime). Motion in the sample is detected between successive images of thesample, where at least part of that motion is generated as a result ofan internal or intrinsic force, such as the pulsatile motion from thecardiovascular system. The next step is correcting the detected motiondue to sources not of interest, or any source that is not the internalor intrinsic force. Motion correction can be accomplished by variousmethods, such as image registration, cross-correlation, orspatio-temporal filtering. This generates detected intrinsic motion invarious successive images of the sample that is due primarily to theinternal or intrinsic force. A next step is determining motioninformation between images of the tissue sample that show detectedintrinsic motion. The motion information can be the optical phasedifference between successive frames or with reference to a specificframe of the images of the sample in which the intrinsic motion has beendetected. For example, a selected frame (x,z) of complex data ismultiplied with complex conjugate of the successive complex data frameafter the previously mentioned motion correction. The angle of resultingcomplex data frame is the optical phase difference. The next step is totranslate the motion information in the images to displacement of thetissue sample over time. The step of calculating displacement based ondetected phase different information may be by phase-sensitivemeasurements, speckle tracking, motion tracking, or digital correlationmethods, and the displacement can be in any direction. For example, thephase difference, Δφ can be directly translated to displacement, Δd by

${\Delta d} = \frac{{\Delta\varphi\lambda}_{0}}{4\pi n}$where λ₀ is the central wavelength of the imaging system and n is therefractive index of the sample at the central wavelength of the imagingsystem. Spatio-temporal smoothing or averaging can be applied at anystep to reduce noise.

A next preferred step is to use the displacement of the tissue sample tocalculate strain. The strain, ∈ can be calculated by least-squareslinear fitting the measured displacement, Δd, over a region of interest,Δz, along a selected axis of deformation by

$\epsilon = {\frac{\Delta d}{\Delta z}.}$A next preferred step is to use the calculated strain to calculateelasticity of the tissue sample either with or without externalmeasurement of the baseline or dynamic pressure. For example, in thecornea, the intraocular pressure can be assumed as the applied pressureso the elasticity, E, by

$E = {\frac{IOP}{\epsilon}.}$In addition a pre-calibrated stress sensor could be utilized, whichcould map the applied stress, σ, to the sample to calculate theelasticity by

$E = {\frac{\sigma}{\epsilon}.}$Different calculated properties, such as displacement, strain andelasticity, may be attributed to different regions of the tissue samplebased on the imaging. The region exhibiting lesser stiffness/elasticityand greater displacement/strain is softer, and the region exhibitinggreater stiffness/elasticity and lesser displacement/strain is stiffer.The step of determining or calculating biomechanical properties of thetissue may be considered the step of calculating one or more ofdisplacement, strain, bulk modulus, and/or Young's modulus using theoptical phase difference information that is detected or determined fromimages of the tissue sample.

In preferred embodiments, the tissue samples are ocular tissue samples.The pressure measured is the eye-globe intraocular pressure.

An example demonstrated herein is a porcine cornea in the wholeeye-globe configuration with an artificially controlled pressure tosimulate the heartbeat.

The exemplary OCE system is phase-sensitive. The OCE system ispreferably used in conjunction with a data analyzing algorithm. Based onmeasurements from the OCE system, the data analyzing algorithm canquantify the mechanical parameters of the cornea. Any suitable computeror data processor programmed with the data analyzing algorithm can beused to make these calculations. The mechanical parameters includeparameters such as displacement amplitude, strain, bulk modulus,elasticity, stiffness, and/or Young's modulus. In preferred embodiments,the data analyzing algorithm comprises the steps of correcting the bulkmotion of the sample, calculating the phase difference, calculating thedisplacement, calculating the strain, mapping an applied stress, andcalculating the elasticity of the sample. A preferred example of a dataanalyzing algorithm that may be useful in the current system and methodis shown in FIG. 2 . These calculations are performed for each step intime and can be correlated to time-matched physiological measurements,such as by an electrocardiogram, pressure transducer, or pulse oximeter.

FIG. 2 shows a schematic of an example of a phase-sensitive OCE system.The setup consists of an optical coherence tomography (OCT) imagingsystem and an artificial intraocular pressure (IOP) control system forvalidation testing. The OCT system is based on a Michelson interferomterand a superluminescent diode (SLD). The light from the SLD is fed to a50/50 fiber optic coupler. The light is them split into two arms. Onearm is the sample arm, which is focused on the sample with an objectivelens. The beam is scanned across the sample by a pair of mirror-mountedgalvanometer mirrors that are controlled by software driving adigital-to-analog converter (DAC). The back scattered light from thesample arm is then collected and combined with the reflected light fromthe reference arm, which has a mirror. Here the interference pattern isthen spread by an optical grating and focused onto a charge-coupleddevice (CDD) line scan camera. A frame grabber in the computer capturesthe data from the camera for processing. The intraocular pressure (IOP)is controlled by a closed-loop feedback system. The closed loop iscomprised of a pressure transducer and a micro-infusion pump which arecontrolled by custom software.

For experimental validation, the biomechanical properties of in situporcine corneas were analyzed using a phase-sensitive OCE system.Experiments were performed on the corneas of pigs in situ (intacteye-globe). The intraocular pressure (IOP) inside the eye-globe wasartificially modulated.

Experimental validation was also performed on anesthetized rabbits invivo. The OCE system and accompanying data analysis algorithm wasdemonstrated as a promising tool for noninvasive assessment of thechanges in the corneal biomechanical properties due to variousphysiological conditions and treatments. The high displacementsensitivity of phase-sensitive OCT detection enables the measurement ofsub-micron displacements throughout the cornea, which is critical for invivo study as the pulsatile motion is very small and undetectable byother means. In addition, the high spatial resolution of OCT allows ahighly-localized investigation of the mechanical properties of thecornea.

EXAMPLE 1. IN SITU MEASUREMENT AND VALIDATION

A phase-sensitive OCE system was utilized, which consisted of aphase-sensitive spectral domain OCT system. The OCT system was comprisedof a superluminescent diode with central wavelength of 840 nm, bandwidthof ˜50 nm, and output power of ˜12 mW. The axial resolution of thesystem was ˜6 μm in air. The experimentally measured displacementstability of the system was ˜5 nm in air.

A validation study was initially conducted on in situ porcine corneas inthe whole eye-globe configuration. The eye-globes were placed in aholder and cannulated with two needles. One needle was used for fluidinfusion and withdrawal, and the other needle was used for pressuresensing for closed-loop feedback. The TOP was then fluctuated in asinusoidal pattern to simulate the intrinsic pulsatile motion induced bythe heartbeat. Repeated OCT images were acquired at the same locationfor a period of a several seconds. After measurements on the corneas,they were then cross-linked by the standard riboflavin/UV “Dresden”corneal collagen crosslinking (CXL) protocol. The OCE measurements werethen repeated.

As shown in FIG. 3 , there was a displacement in cornea in the upwardsdirection (i.e., compression of the tissue) when the fluid was infusedinto the eye-globe (top row). As fluid was withdrawn from the eye-globe(bottom row), the displacement was in the downwards direction (i.e.,expansion in the tissue). Since the data was normalized to the surface,upwards displacement indicates compression, and downwards displacementindicates extension. The amplitude of the displacement was greater inthe untreated corneas (left column) as compared to the crosslinked (CXL)corneas (right column) indicating an increase in stiffness after CXL.

FIG. 4 shows the strain as calculated from FIG. 3 . There is a positivestrain (i.e., compression of the tissue) when fluid is infused into theeye-globe as seen in the top row of FIG. 4 . On the other hand, there isa negative strain (i.e., expansion in the tissue) when fluid iswithdrawn from the eye-globe as seen in the bottom row of FIG. 4 .Crosslinking (CXL) reduces the magnitude of the strain, as seen whencomparing the right column to the left column in FIG. 4 , indicatingthat there was an increase in stiffness after CXL.

FIG. 5 shows a summary over one pulsation cycle showing the averagestrain across the cornea for the untreated (UT, triangle) and CXL(square) cornea as a function of the intraocular pressure (IOP) withinthe eye-globe. The points are the mean values and the error bars are thestandard deviation.

The experiments were repeated on 3 corneas. FIG. 6(a) shows the (points)average and (error bars) standard deviation of the strain in 3 porcinecorneas before (UT, triangle) and after CXL (squares) as a function ofIOP. FIG. 6(b) shows that there was a statistical difference between thestiffness of the porcine corneas (UT) before and (CXL) afterUV/riboflavin crosslinking with the p-value of the two sample t-testindicated.

Additional validation was performed on a partially CXL porcine cornea insitu. Here the same “Dresden” CXL protocol was performed on the cornea,except half of the cornea was covered to prevent any incidentultraviolet illumination, thereby preventing any crosslinking. FIG. 7(a)shows an OCT structural image where the left side was CXL and the rightside was untreated (UT). FIG. 7(b) shows the strain as a function of theeye-globe intraocular pressure (IOP) for the untreated (UT, triangle)and CXL (square) regions of the cornea. The points are the averagestrain, and the error bars are the standard deviation.

EXAMPLE 2. IN VIVO CORNEAL STIFFNESS

To validate the ability of the system and algorithms to measure thepulsatile motion in the cornea in vivo, experiments were carried out ina live rabbit model. The animals were anesthetized with an approved doseand monitored by trained and certified veterinarians. A pressuretransducer was mounted to the chest of the animal for corroboration withthe OCE measurements. A glass slide was mounted to the OCE system, andthe rabbit cornea was lightly compressed against the glass slide forstabilization. The same methodology, as outlined in FIG. 2 was utilized.

FIGS. 8 (a) and (b) show two sample differential displacement frames(i.e., change in displacement from the previous frame) plotted as afunction of the voltage from the chest-mounted pressure transducer. Thevoltage corresponding to the frame is indicated by the vertical line inthe voltage plot. FIG. 8 (c) shows the average (solid line) and standarddeviation (shaded region) of the strain in the cornea, which is plottedalongside the voltage of the chest-mounted pressure transducer. Theresults show the system and method can detect the pulsatile motion inthe cornea due to the heartbeat.

FIG. 9 (a) shows the results of the strain in the rabbit cornea in vivobefore (UT, dashed line) and after (CXL, solid line) riboflavin/UVcrosslinking. The lines are the regional average and the shaded regionis the standard deviation. There was a statistical difference in thestrain after CXL as shown in FIG. 9 (b), indicating a significantincrease in stiffness.

What is claimed is:
 1. A system for quantifying biomechanical propertiesof tissues in a subject, comprising: a phase sensitive low coherenceinterferometry or optical coherence tomography subsystem for imaging thetissues and measuring intrinsic displacements in the tissues resultingfrom a heartbeat of the subject; and a data processor programmed with analgorithm for quantifying biomechanical properties of the tissues basedon the intrinsic displacements.
 2. The system of claim 1 wherein thealgorithm quantifies one or more of phase difference, displacementamplitude, strain, elasticity, bulk modulus, and Young's modulus of thetissues.
 3. The system of claim 2, wherein the algorithm quantifiesdisplacement amplitude, wherein the displacement amplitude is calculatedusing phase-sensitive measurements, speckle tracking, motion tracking,or digital correlation methods, and wherein the displacement amplitudeis in any direction.
 4. The system of claim 1, wherein the dataprocessor corrects the intrinsic displacements to remove motion causedby forces other than the heartbeat before quantifying the biomechanicalproperties of the tissues.
 5. The system of claim 1, wherein the tissuesare ocular tissues.
 6. The system of claim 1, wherein the phasesensitive low coherence interferometry or optical coherence tomographysubsystem images the tissues in 2D, 3D, or 4D.
 7. The system of claim 1,wherein the optical coherence tomography subsystem images the tissues.